Compositions and methods for detection of zika virus

ABSTRACT

Methods for the rapid detection of the presence or absence of Zika virus in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting Zika virus and kits are provided that are designed for the detection of Zika virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 15/456,071, filed Mar. 10, 2017, which claims thebenefit of priority of U.S. Provisional Patent Application Nos.62/306,803, filed Mar. 11, 2016, and 62/327,688, filed Apr. 26, 2016,which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present disclosure relates to the field of viral diagnostics, andmore particularly to detection of Zika virus.

BACKGROUND OF THE INVENTION

Zika virus, a member of the family Flaviviridae and the genusFlavivirus, is a virus that is transmitted by Aedes mosquitoes, whichinclude A. aegypti and A. albopictus. Zika virus is related to denguevirus, yellow fever virus, Japanese encephalitis virus (JEV), and WestNile virus (WNV). Infection by the Zika virus is known as Zika fever,which in humans, may cause fever, rash, and malaise. Zika virusinfection is linked with neurologic conditions in adults, including theGuillain-Barré syndrome. Additionally, as of early 2016, Zika virusinfection in pregnant women has been linked with miscarriage and/ormicrocephaly. To date, there are no known effective drug treatments foror vaccines against Zika virus infection.

In 2013-2014, there were epidemic outbreaks of Zika virus in Oceania. InMay 2015, the first confirmed case of Zika virus infection wasidentified in Brazil. Since May 2015, there have been an estimated440,000 to 1,300,000 people infected in Brazil. The recent outbreaks inBrazil have been linked with the high number of claims ofmicroencephaly. As of early 2016 a widespread outbreak of Zika virus wasongoing, primarily in the Americas, originating from Brazil andspreading to other countries in South America, Central America, Mexico,and the Caribbean.

Serological tests (enzyme-linked immunosorbent assay (ELISA) orimmunofluorescence) for Zika virus have been developed. However,cross-reactivity with other flaviviruses, including dengue or yellowfever, limits the utility of an IgM antibody diagnostic test. Moreover,antibodies may not be present in the early phase of the infection, whichfurther reduces the utility and suitability of a serologic test foracute infection. Thus, there is a need in the art for a quick, reliable,specific, and sensitive method to detect Zika virus.

SUMMARY OF THE INVENTION

Certain embodiments in the present disclosure relate to methods for therapid detection of the presence or absence of Zika virus in a biologicalor non-biological sample, for example, multiplex detection of Zika virusby quantitative real-time reverse-transcriptase polymerase chainreaction (PCR) in a single test tube. Embodiments include methods ofdetection of Zika virus comprising performing at least one cycling step,which may include an amplifying step and a hybridizing step.Furthermore, embodiments include primers, probes, and kits that aredesigned for the detection of Zika virus in a single tube. The detectionmethods are designed to target various regions of the Zika virus genome.For example, the methods are designed to target the region of the genomethat encodes the envelope (E) region, and/or target non-structural (NS)regions, such as NS3, NS4B, and NS5.

In one embodiment, a method for detecting Zika virus in a sample isprovided, comprising performing an amplifying step including contactingthe sample with a set of primers to produce an amplification product ifZika virus is present in the sample; performing a hybridizing stepincluding contacting the amplification product with one or moredetectable probes; and detecting the presence or absence of theamplified product, wherein the presence of the amplified product isindicative of the presence of Zika virus in the sample and wherein theabsence of the amplified product is indicative of the absence of Zikavirus in the sample; wherein the set of primer comprises or consists ofa sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 4,5, 6, 7, and 8, or a complement thereof; and wherein the detectableprobe comprises or consists of a sequence selected from the groupconsisting of SEQ ID NOs:9, 10, 11, and 12, or a complement thereof.

In one embodiment, the primer set for amplification of the Zika virustarget includes a first primer comprising a first oligonucleotidesequence selected from the group consisting of SEQ ID NOs:1, 2, 3, and4, or a complement thereof, and a second primer comprising a secondoligonucleotide sequence selected from the group consisting of SEQ IDNOs:5, 6, 7, and 8, or a complement thereof, and the detectable probefor detection of the amplification product includes the nucleic acidsequences of SEQ ID NOs:9, 10, 11, and 12, or a complement thereof.

Other embodiments provide an oligonucleotide comprising or consisting ofa sequence of nucleotides selected from SEQ ID NOs:1-12, or a complementthereof, which oligonucleotide has 100 or fewer nucleotides. In anotherembodiment, the present disclosure provides an oligonucleotide thatincludes a nucleic acid having at least 70% sequence identity (e.g., atleast 75%, 80%, 85%, 90% or 95%, etc.) to one of SEQ ID NOs:1-12, or acomplement thereof, which oligonucleotide has 100 or fewer nucleotides.Generally, these oligonucleotides may be primer nucleic acids, probenucleic acids, or the like in these embodiments. In certain of theseembodiments, the oligonucleotides have 40 or fewer nucleotides (e.g., 35or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides,20 or fewer nucleotides, 15 or fewer nucleotides, etc.) In someembodiments, the oligonucleotides comprise at least one modifiednucleotide, e.g., to alter nucleic acid hybridization stability relativeto unmodified nucleotides. Optionally, the oligonucleotides comprise atleast one label and optionally at least one quencher moiety. In someembodiments, the oligonucleotides include at least one conservativelymodified variation.

“Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence refers to thosenucleic acids, which encode identical or essentially identical aminoacid sequences, or, where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. One of skill in the artwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single nucleotide or a small percentage ofnucleotides (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ nuclease activity. Thus, the donor fluorescent moiety and theacceptor moiety, e.g., a quencher, may be within no more than 5 to 20nucleotides (e.g., within 8 or 10 nucleotides) of each other along thelength of the probe. In another aspect, the probe includes a nucleicacid sequence that permits secondary structure formation. Such secondarystructure formation may result in spatial proximity between the firstand second fluorescent moiety. According to this method, the secondfluorescent moiety on the probe can be a quencher.

In one aspect, the specific Zika virus probes may be labeled with afluorescent dye which acts as a reporter. The probe may also have asecond dye which acts as a quencher. The reporter dye is measured at adefined wavelength, thus permitting detection and discrimination of theamplified Zika virus target. The fluorescent signal of the intact probesis suppressed by the quencher dye. During the PCR amplification step,hybridization of the probes to the specific single-stranded DNA templateresults in cleavage by the 5′ to 3′ nuclease activity of the DNApolymerase resulting in separation of the reporter and quencher dyes andthe generation of a fluorescent signal. With each PCR cycle, increasingamounts of cleaved probes are generated and the cumulative signal of thereporter dye is concomitantly increased. Optionally, one or moreadditional probes (e.g., such as an internal reference control or othertargeted probe (e.g., other viral nucleic acids) may also be labeledwith a reporter fluorescent dye, unique and distinct from thefluorescent dye label associated with the Zika virus probe. In suchcase, because the specific reporter dyes are measured at definedwavelengths, simultaneous detection and discrimination of the amplifiedZika target and the one or more additional probes is possible.

The present disclosure also provides for methods of detecting thepresence or absence of Zika virus, or Zika virus nucleic acid, in abiological sample from an individual. These methods can be employed todetect the presence or absence of Zika virus or Zika virus nucleic acidin plasma, for use in blood screening and diagnostic testing.Additionally, the same test may be used by someone experienced in theart to assess urine and other sample types to detect Zika virus or Zikavirus nucleic acid. Such methods generally include performing at leastone cycling step, which includes an amplifying step and a dye-bindingstep. Typically, the amplifying step includes contacting the sample witha plurality of pairs of oligonucleotide primers to produce one or moreamplification products if a nucleic acid molecule is present in thesample, and the dye-binding step includes contacting the amplificationproduct with a double-stranded DNA binding dye. Such methods alsoinclude detecting the presence or absence of binding of thedouble-stranded DNA binding dye into the amplification product, whereinthe presence of binding is indicative of the presence of Zika virus orZika virus nucleic acid in the sample, and wherein the absence ofbinding is indicative of the absence of Zika virus or Zika virus nucleicacid in the sample. A representative double-stranded DNA binding dye isethidium bromide. Other nucleic acid-binding dyes include DAPI, Hoechstdyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO®and SYBR® Green. In addition, such methods also can include determiningthe melting temperature between the amplification product and thedouble-stranded DNA binding dye, wherein the melting temperatureconfirms the presence or absence of Zika virus or Zika virus nucleicacid.

In a further embodiment, a kit for detecting one or more nucleic acidsof Zika virus is provided. The kit can include one or more sets ofprimers specific for amplification of the gene target; and one or moredetectable oligonucleotide probes specific for detection of theamplification products.

In one aspect, the kit can include probes already labeled with donor andcorresponding acceptor moieties, e.g., another fluorescent moiety or adark quencher, or can include fluorophoric moieties for labeling theprobes. The kit can also include nucleoside triphosphates, nucleic acidpolymerase, and buffers necessary for the function of the nucleic acidpolymerase. The kit can also include a package insert and instructionsfor using the primers, probes, and fluorophoric moieties to detect thepresence or absence of Zika virus nucleic acid in a sample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present subject matter, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows PCR growth curves of experiments showing that the primersand probes specific for the E, NS3, and NS5 regions of the Zika virusgenome detect Zika virus.

FIG. 2 shows PCR growth curves of experiments showing that the primersand probes specific for the E and NS5 regions of the Zika virus genomedetect Zika virus.

FIG. 3 shows PCR growth curves of experiments, in the presence of aninternal reference control, showing the capacity of the Zika virusprimers and probes for multiplexing with other targets/nucleic acids.

FIG. 4 shows PCR growth curves of experiments showing that the primersand probes detect Zika virus from a biological sample, e.g., plasma.

DETAILED DESCRIPTION OF THE INVENTION

Diagnosis of Zika virus infection by nucleic acid amplification providesa method for rapidly, accurately, reliably, specifically, andsensitively detecting the viral infection. A quantitative real-timereverse-transcriptase PCR assay for detecting Zika virus in anon-biological or biological sample is described herein. Primers andprobes for detecting Zika virus are provided, as are articles ofmanufacture or kits containing such primers and probes. The increasedspecificity and sensitivity of real-time PCR for detection of Zika viruscompared to other methods, as well as the improved features of real-timePCR including sample containment and real-time detection of theamplified product, make feasible the implementation of this technologyfor routine diagnosis of Zika virus infections in the clinicallaboratory. Additionally, this technology may be employed for bloodscreening as well as for prognosis. This Zika virus detection assay mayalso be multiplexed with other assays for the detection of other nucleicacids, e.g., dengue virus, chikungunya virus, and West Nile virus, inparallel.

The Zika virus genome is a positive sense single-stranded RNA molecule10,794 bases long with two non-coding regions known as the 5′NCR and the3′ NCR. The open reading frame of the Zika virus reads5′-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′. It encodes for apolypeptide that is subsequently cleaved into capsid (C), precursormembrane (prM), envelope (E), and non-structural proteins (NS). Theenvelope comprises most of the virion surface and is involved withaspects of replication.

The present disclosure includes oligonucleotide primers and fluorescentlabeled hydrolysis probes that hybridize to the Zika virus genome (e.g.,aa region of the genome that encodes the envelope (E) region, and/ortarget non-structural (NS) regions, such as NS3, NS4B, and NS5), inorder to specifically identify Zika virus using, e.g., TaqMan®amplification and detection technology. The oligonucleotidesspecifically hybridize to the envelope (E) region, and/or targetnon-structural (NS) regions, such as NS3, NS4B, and NS5. Havingoligonucleotides that hybridize to multiple locations in the genome isadvantageous for improved sensitivity compared to targeting a singlecopy genetic locus.

The disclosed methods may include performing at least one cycling stepthat includes amplifying one or more portions of the nucleic acidmolecule gene target from a sample using one or more pairs of primers.“Zika virus primer(s)” as used herein refer to oligonucleotide primersthat specifically anneal to nucleic acid sequences found in the Zikavirus genome, and initiate DNA synthesis therefrom under appropriateconditions producing the respective amplification products. Examples ofnucleic acid sequences found in the Zika virus genome, include nucleicacids within the envelope (E) region, and/or target non-structural (NS)regions, such as NS3, NS4B, and NS5. Each of the discussed Zika virusprimers anneals to a target within the envelope (E) region, and/ortarget non-structural (NS) regions, such as NS3, NS4B, and NS5 such thatat least a portion of each amplification product contains nucleic acidsequence corresponding to the target. The one or more amplificationproducts are produced provided that one or more nucleic acid is presentin the sample, thus the presence of the one or more amplificationproducts is indicative of the presence of Zika virus in the sample. Theamplification product should contain the nucleic acid sequences that arecomplementary to one or more detectable probes for Zika virus. “Zikavirus probe(s)” as used herein refer to oligonucleotide probes thatspecifically anneal to nucleic acid sequences found in the Zika virusgenome. Each cycling step includes an amplification step, ahybridization step, and a detection step, in which the sample iscontacted with the one or more detectable Zika virus probes fordetection of the presence or absence of Zika virus in the sample.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., nucleic acidmolecules from the Zika virus genome). Amplifying a nucleic acidmolecule typically includes denaturing the template nucleic acid,annealing primers to the template nucleic acid at a temperature that isbelow the melting temperatures of the primers, and enzymaticallyelongating from the primers to generate an amplification product.Amplification typically requires the presence of deoxyribonucleosidetriphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and anappropriate buffer and/or co-factors for optimal activity of thepolymerase enzyme (e.g., MgCl₂ and/or KCl).

The term “primer” as used herein is known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′-OH group where further “nucleotides”may be attached by a template-dependent DNA polymerase establishing 3′to 5′ phosphodiester linkage whereby deoxynucleoside triphosphates areused and whereby pyrophosphate is released.

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. “Hybridization conditions” typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished, if necessary.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., at-butyl benzyl, a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, aC5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, aC7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, aC5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl ribo-U, 2′-0-methyl ribo-C,an N4-ethyl-dC, an N6-methyl-dA, and the like. Many other modifiednucleotides that can be substituted in the oligonucleotides are referredto herein or are otherwise known in the art. In certain embodiments,modified nucleotide substitutions modify melting temperatures (Tm) ofthe oligonucleotides relative to the melting temperatures ofcorresponding unmodified oligonucleotides. To further illustrate,certain modified nucleotide substitutions can reduce non-specificnucleic acid amplification (e.g., minimize primer dimer formation or thelike), increase the yield of an intended target amplicon, and/or thelike in some embodiments. Examples of these types of nucleic acidmodifications are described in, e.g., U.S. Pat. No. 6,001,611, which isincorporated herein by reference. Other modified nucleotidesubstitutions may alter the stability of the oligonucleotide, or provideother desirable features.

Detection of Zika Virus

The present disclosure provides methods to detect Zika virus byamplifying, for example, a portion of the Zika virus nucleic acidsequence. Nucleic acid sequences of Zika virus are available (e.g.,Reference Strain: Uganda 1957, Accession: NC_012532). Specifically,primers and probes to amplify and detect Zika virus nucleic acidmolecule targets are provided by the embodiments in the presentdisclosure.

For detection of Zika virus, primers and probes to amplify the Zikavirus are provided. Zika virus nucleic acids other than thoseexemplified herein can also be used to detect Zika virus in a sample.For example, functional variants can be evaluated for specificity and/orsensitivity by those of skill in the art using routine methods.Representative functional variants can include, e.g., one or moredeletions, insertions, and/or substitutions in the Zika virus nucleicacids disclosed herein.

More specifically, embodiments of the oligonucleotides each include anucleic acid with a sequence selected from SEQ ID NOs:1-12, asubstantially identical variant thereof in which the variant has atleast, e.g., 80%, 90%, or 95% sequence identity to one of SEQ IDNOs:1-12, or a complement of SEQ ID NOs:1-12 and the variant.

TABLE 1 Zika Virus Forward Primers Forward Primers SEQ ID Oligo Name NO:Sequence Modifications ZIKF1151_21TBB 1 AACATGGCGGAGGTAAGATCJJ = t-butylbenzyl dC ZIKF9806_20TBB 2 CTCCCACCACTTCAACAAGJJ = t-butylbenzyl dC ZIKF6006_21TBB 3 GAACCCCAACAAACCTGGAGJJ = t-butylbenzyl dA ZIKF7369_26TBB 4 TTGTGGATGGAATAGTGGTAACTGJ = t-butylbenzyl dC AJ

TABLE 2 Zika Virus Reverse Primers Reverse Primers SEQ ID Oligo Name NO:Sequence Modifications ZIKR1236_30TBB 5 TAATGTTCTTTT J = GCAGACATATTGt-butylbenzyl dC AGTGTJ ZIKR9896_19_TBB 6 CAGTCTCCCGGA J = TGCTCCJt-butylbenzyl dA ZIKR6075_26TBB 7 CTGGAGGTAAAT J = GTTGTCAAGAAGt-butylbenzyl dA CJ ZIKR7470_17_TBB 8 CJAGCCTCCCCC J = CATCCtbutylbenzyl dC

TABLE 3 Zika Virus Probes Probes SEQ ID Oligo Name NO: SequenceModifications ZIKPS1197_35HQ8 9 HTGCCCAACQACA P = phosphate,AGGTGAAGCCTAC H = HEX-Thr, CTTGACAAGCAP Q = BHQ2 ZIKPS9844_27HQ8 10HCCGCCACCQAAG P = phosphate, ATGAACTGATTGG H = HEX-Thr, CCGP Q = BHQ2ZIKPS6019_33HQ8 11 HTGGAGGTGQGGT P = phosphate, GTGCAGAGACTGAH = HEX-Thr, TGAAGACCAP Q = BHQ2 ZIKPS7372_42HQ8 12 HTGACACAAQTGAP = phosphate, CAATTGACCCCCA H = HEX-Thr, AGTGGAGAAGAAG Q = BHQ2 ATGGGP

In one embodiment, the above described sets of Zika virus primers andprobes are used in order to provide for detection of Zika virus in abiological sample suspected of containing Zika virus (Tables 1-3). Thesets of primers and probes may comprise or consist of the primers andprobes specific for the Zika virus nucleic acid sequences, comprising orconsisting of the nucleic acid sequences of SEQ ID NOs:1-12. In anotherembodiment, the primers and probes for the Zika virus target comprise orconsist of a functionally active variant of any of the primers andprobes of SEQ ID NOs:1-12.

A functionally active variant of any of the primers and/or probes of SEQID NOs:1-12 may be identified by using the primers and/or probes in thedisclosed methods. A functionally active variant of a primer and/orprobe of any of the SEQ ID NOs:1-12 pertains to a primer and/or probewhich provide a similar or higher specificity and sensitivity in thedescribed method or kit as compared to the respective sequence of SEQ IDNOs:1-12.

The variant may, e.g., vary from the sequence of SEQ ID NOs:1-12 by oneor more nucleotide additions, deletions or substitutions such as one ormore nucleotide additions, deletions or substitutions at the 5′ endand/or the 3′ end of the respective sequence of SEQ ID NOs:1-12. Asdetailed above, a primer (and/or probe) may be chemically modified,i.e., a primer and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A probe (or a primer) is then a modifiedoligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differfrom a natural “nucleotide” by some modification but still consist of abase or base-like compound, a pentofuranosyl sugar or a pentofuranosylsugar-like compound, a phosphate portion or phosphate-like portion, orcombinations thereof. For example, a “label” may be attached to the baseportion of a “nucleotide” whereby a “modified nucleotide” is obtained. Anatural base in a “nucleotide” may also be replaced by, e.g., a7-desazapurine whereby a “modified nucleotide” is obtained as well. Theterms “modified nucleotide” or “nucleotide analog” are usedinterchangeably in the present application. A “modified nucleoside” (or“nucleoside analog”) differs from a natural nucleoside by somemodification in the manner as outlined above for a “modified nucleotide”(or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify a nucleic acid molecule encoding the Zika virustarget, e.g., nucleic acids encoding alternative portions of Zika viruscan be designed using, for example, a computer program such as OLIGO(Molecular Biology Insights Inc., Cascade, Colo.). Important featureswhen designing oligonucleotides to be used as amplification primersinclude, but are not limited to, an appropriate size amplificationproduct to facilitate detection (e.g., by electrophoresis), similarmelting temperatures for the members of a pair of primers, and thelength of each primer (i.e., the primers need to be long enough toanneal with sequence-specificity and to initiate synthesis but not solong that fidelity is reduced during oligonucleotide synthesis).Typically, oligonucleotide primers are 8 to 50 nucleotides in length(e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, or 50 nucleotides in length).

In addition to a set of primers, the methods may use one or more probesin order to detect the presence or absence of Zika virus. The term“probe” refers to synthetically or biologically produced nucleic acids(DNA or RNA), which by design or selection, contain specific nucleotidesequences that allow them to hybridize under defined predeterminedstringencies specifically (i.e., preferentially) to “target nucleicacids”, in the present case to a Zika virus (target) nucleic acid. A“probe” can be referred to as a “detection probe” meaning that itdetects the target nucleic acid.

In some embodiments, the described Zika virus probes can be labeled withat least one fluorescent label. In one embodiment, the Zika virus probescan be labeled with a donor fluorescent moiety, e.g., a fluorescent dye,and a corresponding acceptor moiety, e.g., a quencher.

In one embodiment, the probe comprises or consists of a fluorescentmoiety and the nucleic acid sequences comprise or consist of SEQ IDNOs:9-12.

Designing oligonucleotides to be used as probes can be performed in amanner similar to the design of primers. Embodiments may use a singleprobe or a pair of probes for detection of the amplification product.Depending on the embodiment, the probe(s) use may comprise at least onelabel and/or at least one quencher moiety. As with the primers, theprobes usually have similar melting temperatures, and the length of eachprobe must be sufficient for sequence-specific hybridization to occurbut not so long that fidelity is reduced during synthesis.Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22,23, 24, or 25) nucleotides in length.

Constructs can include vectors each containing one of Zika virus primersand probes nucleic acid molecules (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6,7, 8, 9, and 10). Constructs can be used, for example, as controltemplate nucleic acid molecules. Vectors suitable for use arecommercially available and/or produced by recombinant nucleic acidtechnology methods routine in the art. Zika virus nucleic acid moleculescan be obtained, for example, by chemical synthesis, direct cloning fromZika virus, or by nucleic acid amplification.

Constructs suitable for use in the methods typically include, inaddition to the Zika virus nucleic acid molecules (e.g., a nucleic acidmolecule that contains one or more sequences of SEQ ID NOs:1-12),sequences encoding a selectable marker (e.g., an antibiotic resistancegene) for selecting desired constructs and/or transformants, and anorigin of replication. The choice of vector systems usually depends uponseveral factors, including, but not limited to, the choice of hostcells, replication efficiency, selectability, inducibility, and the easeof recovery.

Constructs containing Zika virus nucleic acid molecules can bepropagated in a host cell. As used herein, the term host cell is meantto include prokaryotes and eukaryotes such as yeast, plant and animalcells. Prokaryotic hosts may include E. coli, Salmonella typhimurium,Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts includeyeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cellssuch as COS cells or Chinese hamster ovary (CHO) cells, insect cells,and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. Aconstruct can be introduced into a host cell using any of the techniquescommonly known to those of ordinary skill in the art. For example,calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transferare common methods for introducing nucleic acids into host cells. Inaddition, naked DNA can be delivered directly to cells (see, e.g., U.S.Pat. Nos. 5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in some embodiments include oligonucleotidescapable of acting as points of initiation of nucleic acid synthesiswithin the described Zika virus nucleic acid sequences (e.g., SEQ IDNOs: 1-8). A primer can be purified from a restriction digest byconventional methods, or it can be produced synthetically. The primer ispreferably single-stranded for maximum efficiency in amplification, butthe primer can be double-stranded. Double-stranded primers are firstdenatured, i.e., treated to separate the strands. One method ofdenaturing double stranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence. The temperature forannealing is usually from about 35° C. to about 65° C. (e.g., about 40°C. to about 60° C.; about 45° C. to about 50° C.). Annealing times canbe from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec;about 30 sec to about 40 sec). The reaction mixture is then adjusted toa temperature at which the activity of the polymerase is promoted oroptimized, i.e., a temperature sufficient for extension to occur fromthe annealed primer to generate products complementary to the templatenucleic acid. The temperature should be sufficient to synthesize anextension product from each primer that is annealed to a nucleic acidtemplate, but should not be so high as to denature an extension productfrom its complementary template (e.g., the temperature for extensiongenerally ranges from about 40° C. to about 80° C. (e.g., about 50° C.to about 70° C.; about 60° C.). Extension times can be from about 10 secto about 5 min (e.g., about 30 sec to about 4 min; about 1 min to about3 min; about 1 min 30 sec to about 2 min).

The genome of a retrovirus or RNA virus, such as Zika virus as well asother flaviviruses, is comprised of a ribonucleic acid, i.e., RNA. Insuch case, the template nucleic acid, RNA, must first be transcribedinto complementary DNA (cDNA) via the action of the enzyme reversetranscriptase. Reverse transcriptases use an RNA template and a shortprimer complementary to the 3′ end of the RNA to direct synthesis of thefirst strand cDNA, which can then be used directly as a template forpolymerase chain reaction.

PCR assays can employ Zika virus nucleic acid such as RNA or DNA (cDNA).The template nucleic acid need not be purified; it may be a minorfraction of a complex mixture, such as Zika virus nucleic acid containedin human cells. Zika virus nucleic acid molecules may be extracted froma biological sample by routine techniques such as those described inDiagnostic Molecular Microbiology: Principles and Applications (Persinget al. (eds), 1993, American Society for Microbiology, Washington D.C.).Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

The oligonudeotide primers (e.g., SEQ ID NOs:1-8) are combined with PCRreagents under reaction conditions that induce primer extension. Forexample, chain extension reactions generally include 50 mM KCl, 10 mMTris-HCl (pH 8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 lagdenatured template DNA, 50 pmoles of each oligonudeotide primer, 2.5 Uof Taq polymerase, and 10% DMSO). The reactions usually contain 150 to320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly-synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target Zika virus nucleic acid molecules. The limiting factors inthe reaction are the amounts of primers, thermostable enzyme, andnucleoside triphosphates present in the reaction. The cycling steps(i.e., denaturation, annealing, and extension) are preferably repeatedat least once. For use in detection, the number of cycling steps willdepend, e.g., on the nature of the sample. If the sample is a complexmixture of nucleic acids, more cycling steps will be required to amplifythe target sequence sufficient for detection. Generally, the cyclingsteps are repeated at least about 20 times, but may be repeated as manyas 40, 60, or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength. In certain systems, non-fluorescent energy can betransferred between donor and acceptor moieties, by way of biomoleculesthat include substantially non-fluorescent donor moieties (see, forexample, U.S. Pat. No. 7,741,467).

In one example, an oligonucleotide probe can contain a donor fluorescentmoiety (e.g., HEX) and a corresponding quencher (e.g., BlackHoleQuenchers™ (BHQ)), which may or not be fluorescent, and which dissipatesthe transferred energy in a form other than light. When the probe isintact, energy transfer typically occurs between the donor and acceptormoieties such that fluorescent emission from the donor fluorescentmoiety is quenched the acceptor moiety. During an extension step of apolymerase chain reaction, a probe bound to an amplification product iscleaved by the 5′ to 3′ nuclease activity of, e.g., a Taq Polymerasesuch that the fluorescent emission of the donor fluorescent moiety is nolonger quenched. Exemplary probes for this purpose are described in,e.g., U.S. Pat. Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly useddonor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchersare DABCYL and TAMRA. Commonly used dark quenchers include BlackHoleQuenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), IowaBlack™, (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry™Quencher 650 (BBQ-650), (Berry & Assoc., Dexter, Mich.).

In another example, two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the Zika virus target nucleic acid sequence.Upon hybridization of the oligonucleotide probes to the amplificationproduct nucleic acid at the appropriate positions, a FRET signal isgenerated. Hybridization temperatures can range from about 35° C. toabout 65° C. for about 10 sec to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorimeter. Excitation to initiate energy transfer, or to allow directdetection of a fluorophore, can be carried out with an argon ion laser,a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber opticlight source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptor moieties“corresponding” refers to an acceptor fluorescent moiety or a darkquencher having an absorbance spectrum that overlaps the emissionspectrum of the donor fluorescent moiety. The wavelength maximum of theemission spectrum of the acceptor fluorescent moiety should be at least100 nm greater than the wavelength maximum of the excitation spectrum ofthe donor fluorescent moiety. Accordingly, efficient non-radiativeenergy transfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Foerster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,helium-cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm can be the distance in Angstroms (Å) from the nucleotide baseto the fluorescent moiety. In general, a linker arm is from about 10 Åto about 25 Å. The linker arm may be of the kind described in WO84/03285. WO 84/03285 also discloses methods for attaching linker armsto a particular nucleotide base, and also for attaching fluorescentmoieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide that contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640-labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of Zika Virus

The present disclosure provides methods for detecting the presence orabsence of Zika virus in a biological or non-biological sample. Methodsprovided avoid problems of sample contamination, false negatives, andfalse positives. The methods include performing at least one cyclingstep that includes amplifying a portion of Zika virus target nucleicacid molecules from a sample using one or more pairs of Zika virusprimers, and a FRET detecting step. Multiple cycling steps areperformed, preferably in a thermocycler. Methods can be performed usingthe Zika virus primers and probes to detect the presence of Zika virus,and the detection of Zika virus indicates the presence of Zika virus inthe sample.

As described herein, amplification products can be detected usinglabeled hybridization probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof Zika virus. TaqMan® technology utilizes one single-strandedhybridization probe labeled with, e.g., one fluorescent dye (e.g., HEX)and one quencher (e.g., BHQ), which may or may not be fluorescent. Whena first fluorescent moiety is excited with light of a suitablewavelength, the absorbed energy is transferred to a second fluorescentmoiety or a dark quencher according to the principles of FRET. Thesecond moiety is generally a quencher molecule. During the annealingstep of the PCR reaction, the labeled hybridization probe binds to thetarget DNA (i.e., the amplification product) and is degraded by the 5′to 3′ nuclease activity of, e.g., the Taq Polymerase during thesubsequent elongation phase. As a result, the fluorescent moiety and thequencher moiety become spatially separated from one another. As aconsequence, upon excitation of the first fluorescent moiety in theabsence of the quencher, the fluorescence emission from the firstfluorescent moiety can be detected. By way of example, an ABI PRISM®7700 Sequence Detection System (Applied Biosystems) uses TaqMan®technology, and is suitable for performing the methods described hereinfor detecting the presence or absence of Zika virus in the sample.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time PCRmethods. Molecular beacon technology uses a hybridization probe labeledwith a first fluorescent moiety and a second fluorescent moiety. Thesecond fluorescent moiety is generally a quencher, and the fluorescentlabels are typically located at each end of the probe. Molecular beacontechnology uses a probe oligonucleotide having sequences that permitsecondary structure formation (e.g., a hairpin). As a result ofsecondary structure formation within the probe, both fluorescentmoieties are in spatial proximity when the probe is in solution. Afterhybridization to the target nucleic acids (i.e., amplificationproducts), the secondary structure of the probe is disrupted and thefluorescent moieties become separated from one another such that afterexcitation with light of a suitable wavelength, the emission of thefirst fluorescent moiety can be detected.

Another common format of FRET technology utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand are generally designed to hybridize in dose proximity to each otherin a target DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler® Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler® instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of Zika virus genomes). If amplification ofZika virus target nucleic acid occurs and an amplification product isproduced, the step of hybridizing results in a detectable signal basedupon FRET between the members of the pair of probes.

Generally, the presence of FRET indicates the presence of Zika virus inthe sample, and the absence of FRET indicates the absence of Zika virusin the sample. Inadequate specimen collection, transportation delays,inappropriate transportation conditions, or use of certain collectionswabs (calcium alginate or aluminum shaft) are all conditions that canaffect the success and/or accuracy of a test result, however.

Representative biological samples that can be used in practicing themethods include, but are not limited to respiratory specimens, urine,fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs,wound swabs, blood cultures, skin, and soft tissue infections.Collection and storage methods of biological samples are known to thoseof skill in the art. Biological samples can be processed (e.g., bynucleic acid extraction methods and/or kits known in the art) to releaseZika virus nucleic acid or in some cases, the biological sample can becontacted directly with the PCR reaction components and the appropriateoligonucleotides.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the Zika virus probes from the Zika virusamplification products can confirm the presence or absence of Zika virusin the sample.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduceor eliminate contamination between one thermocycler run and the next.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods. In one embodiment, a LightCycler® instrument isused. The following patent applications describe real-time PCR as usedin the LightCycler® technology: WO 97/46707, WO 97/46714, and WO97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

One of skill in the art would appreciate that other nucleic acid- orsignal-amplification methods may also be employed. Examples of suchmethods include, without limitation, branched DNA signal amplification,loop-mediated isothermal amplification (LAMP), nucleic acidsequence-based amplification (NASBA), self-sustained sequencereplication (3SR), strand displacement amplification (SDA), or smartamplification process version 2 (SMAP 2).

It is understood that the embodiments of the present disclosure are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

Embodiments of the present disclosure further provide for articles ofmanufacture or kits to detect Zika virus. An article of manufacture caninclude primers and probes used to detect the Zika virus gene target,together with suitable packaging materials. Representative primers andprobes for detection of Zika virus are capable of hybridizing to Zikavirus target nucleic acid molecules. In addition, the kits may alsoinclude suitably packaged reagents and materials needed for DNAimmobilization, hybridization, and detection, such solid supports,buffers, enzymes, and DNA standards. Methods of designing primers andprobes are disclosed herein, and representative examples of primers andprobes that amplify and hybridize to Zika virus target nucleic acidmolecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling theZika virus probes. Examples of suitable FRET donor fluorescent moietiesand corresponding acceptor fluorescent moieties are provided above.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the Zika virus primers andprobes to detect Zika virus in a sample. Articles of manufacture mayadditionally include reagents for carrying out the methods disclosedherein (e.g., buffers, polymerase enzymes, co-factors, or agents toprevent contamination). Such reagents may be specific for one of thecommercially available instruments described herein.

Embodiments of the present disclosure will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples and figures are provided to aid the understandingof the subject matter, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

The targeted region of the Zika virus genome included both structuralregions (e.g., Envelope (E)) as well as non-structural regions (e.g.,NS3, NS4B, and NS5). All nucleic acid sequences were aligned and allprimers and probes were considered and scored for their predictedinclusivity for all known Zika isolates and other properties. Positivecontrols were created by considering the sequences of viruses incirculation. Sequences from publicly available databases were exportedand sequence alignments were performed. The sequences were then filteredto consider contemporary sequences from 2007 on onward. A cDNA done wasfabricated to serve as a positive DNA or RNA control. The cDNA done wasbased on the Suriname 2015 strain relative to the reference strain(African lineage, Uganda), and a historical African strain. Thesequences were aligned relative to the primers and probes and slightadjustments were made to the sequences.

Example 1: Detection of Cultured Zika Virus

RNA from cultured MR 766 Zika virus was extracted using QIAmp Viral RNAMini Kit (Qiagen). Total RNA was quantitated using RiboGreen Assay(ThermoFisher Scientific), Qubit (ThermoFisher Scientific), and NanoDrop(ThermoFisher Scientific). Quantitative real-time RT-PCR was run usingprimers and probes targeting the E, NS3, and NS5 regions at varyingconcentrations of Zika RNA (i.e., 2.5×10⁴ and 2.5×10⁶ copies of RNA/ml).Results show that the primers and probes are able to detect the Eregions (SEQ ID NOs:1, 5, and 9), NS3 (SEQ ID NOs:3, 7, and 11), and NS5(SEQ ID NOs:2, 6, and 10) (see FIG. 1). Further studies were conductedon a wider range of Zika RNA starting concentrations (from 2.5×10⁰,2.5×10¹, 2.5×10², 2.5×10³, 2.5×10⁴, and 2.5×10⁵ copies of RNA/ml),showing that primers and probes were able to detect the E and NS5regions of Zika virus in a dose-dependent fashion (see FIG. 2).Additional assays targeting the E region, in the presence of an internalreference control set of primers/probes, demonstrate the capacity of theZika virus primers and probes to detect Zika virus and to be multiplexedwith other assays for detection (see FIG. 3).

Example 2: Detection of Zika Virus in Biological Samples

In order to show that the primers and probes detecting Zika virus wereable to detect Zika virus from biological samples, e.g., bodily fluids,the primers and probes targeting the E and NS5 regions of the Zika virusgenome were tested against Zika virus RNA obtained from plasma samples.Results show that the Zika virus primers and probes were able to detectZika virus in plasma with specificity and sensitivity. This full processwas conducted several times and the Zika primers and probes were shownto reproducibly detect Zika virus (a representative example is shown inFIG. 4). These results demonstrate that the Zika virus primers andprobes detect the presence of Zika virus in biological samples, e.g.,plasma.

Example 3: Assay Performance of a Zika Nucleic Acid Test for BloodScreening

Human infection with the Zika virus (ZIKV) is associated withmicrocephaly and neurologic disease. Since most infections areasymptomatic, blood donors may unknowingly harbor infection. ZIKVnucleic acid was detected in 2.8% of blood donors during the FrenchPolynesia outbreak in 2013-2014, and cases of transfusion-transmissionhave recently been reported in Brazil. The U.S. FDA issuedrecommendations to reduce the risk of transfusion transmitted ZIKV. Inareas of active transmission, blood establishments must cease bloodcollections unless donations are screened with a nucleic acid test (NAT)for ZIKV or components are pathogen reduced (PR). Currently, PR islimited to platelets and plasma products.

In response to the FDA recommendations, the cobas® Zika test, aqualitative method for detection of ZIKV RNA using polymerase chainreaction (PCR), was developed for use on the cobas® 6800/8800 Systems.These systems incorporate fully-automated sample preparation,amplification and detection, and ready-to-use reagents and controls. Thetest is intended to detect ZIKV RNA in plasma specimens from donors ofwhole blood and blood components, as well as other living donors.

The cobas® Zika test was designed to detect all ZIKV lineages. Mostperformance studies used ZIKV culture supernatant with titer assigned incopies/mL using ZIKV RNA transcript. Studies included specificity,sensitivity, limit of detection (LoD), interfering substances, matrixequivalency, and cross-reactivity. LoD and repeatability were assessedusing 190 replicates across multiple concentrations, reagent lots, anddays. Clinical sensitivity was evaluated by testing 25 samples confirmedZIKV positive using published oligonucleotide sequences (Lanciotti,Emerg Infect Dis, 2008). In addition, 5 clinical specimens were spikedinto negative pooled plasma to create 250 contrived samples at variousdilutions.

The 95% Probit LoD for cobas® Zika was 8.1 copies/mL, and the resultswere reproducible across multiple parameters. Specificity was 100% in500 samples collected in a ZIKV non-endemic region. Neither ZIKVdetectability nor specificity was impacted by potentially-interferingsubstances including albumin, bilirubin, hemoglobin, human DNA andtriglycerides or with various anticoagulants. Human ImmunodeficiencyVirus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV),Chikungunya (CHIKV), Dengue Virus (DENV) serotypes 1-4, and West NileVirus (WNV) did not cross-react. Clinical sensitivity of cobas® Zika was100% with 25 confirmed ZIKV positive samples from 4 different countries.Reactivity was 100% with 250 contrived samples with a range of viralconcentrations.

Example 4: Detection of Zika Virus in Puerto Rican Blood Donors

The U.S. FDA issued recommendations to reduce the risk oftransfusion-transmitted Zika virus (ZIKV), including cessation of bloodcollections in areas of active ZIKV transmission, unless donations arescreened with a ZIKV nucleic acid test (NAT) or are pathogen reduced(PR). Puerto Rico was required to discontinue collections, as noFDA-approved NAT test was available and PR is available only for plasmaand platelet products. In response to FDA recommendations, a qualitativePCR NAT assay was developed to detect ZIKV RNA in plasma from donors ofwhole blood and blood components.

The cobas® Zika test for use on the cobas® 6800/8800 Systems wasdeveloped to detect all ZIKV lineages. Performance was established withstudies including specificity, sensitivity, limit of detection (LoD),potential interfering substances, matrix equivalency, and crossreactivity. The FDA approved use of the test as an Investigational NewDrug (IND). Testing of individual blood donor (IDT) samples from PuertoRico were then begun. Reactive index donations were retested insimulated mini-pools (MP) of 6. Reactive index donations were alsotested by a reference laboratory using a modified CDC PCR ZIKV assay,estimated viral load (VL), ZIKV IgM and IgG antibodies, and plaquereduction neutralization test (PRNT) for IgM reactive samples. Donorswith reactive results were invited to enroll in a follow-up study.

3,573 donations have been screened by cobas® Zika, and 13 (0.36%) testedreactive. Additional results for 9 of these donations are shown on Table4.

TABLE 4 Summary of Testing Results on Donations Reactive with cobas ®Zika cobas ® ZIKA Sample Simulated Modified CDC Serology ID IDT MP PCRMean VL IgG IgM PRNT 1 R R Equivocal 4.20E+02 Negative Negative N/A 2 RR Positive 2.70E+03 Positive Positive Pending 3 R R Positive 5.90E+06Negative Negative N/A 4 R R Positive 7.90E+03 Negative Negative N/A 5 RR Positive 5.40E+04 Negative Negative N/A 6 R NR Positive 3.20E+02Inconclusive Equivocal N/A 7 R R Positive 4.90E+05 Negative Negative N/A8 R R Positive 7.10E+03 Negative Negative N/A 9 R R Positive 7.60E+04Equivocal Negative N/A R Reactive; NR Non-Reactive; N/A Not applicable

These data show that the cobas® Zika test successfully identifiedZIKV-positive donations, allowing for resumption of collections inPuerto Rico.

Example 5: Alternative Assay for Identifying Zika Nucleic Acid inSpecimens

The cobas® Zika test for use on the cobas® 6800/8800 systems wasdeveloped for blood screening under Investigational New Drug (IND). Toestablish sensitivity of the IND test, the FDA required analysis of Zikavirus (ZIKV) positive specimens. Procurement of ZIKV positive specimenswas challenging due to high demand, scarcity of ZIKV confirmedspecimens, and barriers to shipment of specimens from some Zika endemiccountries. A second ZIKV PCR assay was developed as a tool for screeningspecimens with unknown status. This second ZIKV PCR assay specificallyuses the cobas omni Utility Channel, which is ideal for rapidprototyping and high throughput screening. The cobas omni UtilityChannel utilizes most of the reagents used for cobas® IVD tests, exceptthe assay specific primers and probes, allowing for the flexibility toperform laboratory developed tests on the platform.

The second ZIKV PCR assay that targeted a gene locus separate from genelocus targeted by the cobas® Zika test was developed using the cobasomni Utility Channel functionality of the cobas® 6800/8800 system. Thisassay was used to identify “possible” ZIKV specimens by screeningsamples collected in Colombia and El Salvador. Specimens were originallyobtained from chikungunya and dengue endemic areas and found to benegative for those viruses. For screening, due to limited volumes,specimens were diluted to at least 1:3.75 and tested singly. Broad Ctacceptance criteria were used to identify “possible” ZIKV positivespecimens. Specimens identified as “possible” ZIKV by this second assaywere further tested in 4 replicates using RT-PCR with published CDColigonucleotide sequences (Lanciotti, 2008) and once by the cobas® Zikatest. Specimens were considered to be confirmed for ZIKV if consistentreactivity (4/4 replicates) was observed using the CDC oligonudeotides.

Of 1,296 specimens screened by the in-house assay on the cobas omniUtility Channel, 111 were identified as “possible” ZIKV positive.Thirty-seven specimens with sufficient volume were tested using the CDColigos and the cobas® Zika test. Three additional specimens with limitedvolume were diluted 1:100 and tested. Of these 40 “possible” positives,23 specimens, including the 3 diluted specimens, were confirmed positiveby the CDC oligonudeotides and all were reactive with cobas® Zika.Seventeen specimens had <4/4 reactive results with the CDColigonucleotides; 15/17 tested positive with the cobas® Zika test. Theresults are shown on Table 5.

TABLE 5 cobas ® Zika detection in specimens with different levels of CDCreactivity Number of Number of CDC Individual positive ZIKV ResultNumber of reactive Specimens replicates per interpretation specimensusing N = 40 specimen w/CDC oligos cobas ® Zika 23 4 of 4 replicatesConfirmed 23/23 reactive 4 3 of 4 replicates Equivocal  4/4 reactive 1 2of 4 replicates Equivocal  1/1 reactive 7 1 of 4 replicates Equivocal 6/7 reactive 5 0 of 4 replicates Negative  4/5 reactive

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed:
 1. A kit for detecting a nucleic acid sequence of Zikavirus in a sample, the kit comprising: one or more first primers,wherein the one or more first primers consists of an oligonucleotidesequence consisting of SEQ ID NO:1; one or more second primers, whereinthe one or more second primers consists of an oligonucleotide sequenceconsisting of SEQ ID NO:5; and one or more detectable probes, whereinthe one or more detectable probes consists of: (i) an oligonucleotidesequence consisting of SEQ ID NO:9, (ii) a donor fluorescent moiety, and(iii) a corresponding acceptor moiety, and wherein the one or moredetectable probes is configured to hybridize to an amplicon generated bythe one or more first primers and the one or more second primers.
 2. Thekit of claim 1, wherein the acceptor moiety is a quencher.
 3. The kit ofclaim 1, further comprising a primer set and one or more detectableprobes for detecting a nucleic acid sequence from one or more viruses,in parallel.
 4. The kit of claim 3, wherein the one or more otherviruses is selected from the group consisting of dengue virus,chikungunya virus, and West Nile virus.
 5. The kit of claim 1, furthercomprising nucleotide triphosphates, nucleic acid polymerase, andbuffers necessary for the function of the nucleic acid polymerase. 6.The kit of claim 1, wherein at least one of the one or more firstprimers, the one or more second primers, and the one or more detectableprobes comprises at least one modified nucleotide.
 7. A set of primersand one or more detectable probes for the detection of Zika virus in asample, wherein the set of primers consists of: (a) one or more firstprimers, wherein the one or more first primers consists of anoligonucleotide sequence consisting of SEQ ID NO:1; and (b) one or moresecond primers, wherein the one or more second primers consists of anoligonucleotide sequence consisting of SEQ ID NO:5; and wherein the oneor more detectable probes consists of: (i) an oligonucleotide sequenceconsisting of SEQ ID NO:9, (ii) a donor fluorescent moiety, and (iii) acorresponding acceptor moiety.
 8. The set of primers and the one or moredetectable probes of claim 7, wherein the donor fluorescent moiety andthe corresponding acceptor moiety are within no more than 8-20nucleotides of each other on the one or more detectable probes.
 9. Theset of primers and the one or more detectable probes of claim 7, whereinthe acceptor moiety is a quencher.